Book/Report

FZJ-2019-04470

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Charakterisierung des Komplexes aus Na$^{+}$/Ca$^{2+}$-K$^{+}$-Austauscher und cGMP-gesteuertem Kanal in der Plasmamembran von Stäbchenaussensegmenten - biochemische Untersuchungen zur Ultrastruktur Ca$^{2+}$-transportierender Proteine in Photorezeptoren von Vertebraten

Schwarzer, A. (Corresponding author)FZJ*

1998
Forschungszentrum Jülich, Zentralbibliothek, Verlag Jülich

Please use a persistent id in citations: http://hdl.handle.net/2128/22619

Report No.: Juel-3574

Abstract: In vertebrate photoreceptors, the intracellular Ca$^{2+}$ concentration plays a crucial role for signal transduction and light adaption. In bovine rod outer semnents (ROS) intracellular Ca$^{2+}$ concentration is set by Ca$^{2+}$ influx through cGMP-gated channels and extrusion of Ca$^{2+}$ ions by Na$^{+}$/Ca$^{2+}$-K$^{+}$ exchangers. Both proteins are localized in the plasma membrane of the outer segment. In this study the oligomeric state of the Na$^{+}$/Ca$^{2+}$-K$^{+}$ exchanger and its association with the cGMP-gated channel in bovine ROS were investigated, mainly using chemical cross-linking techniques. Proteins were separated by SDS gel electrophoresis and analyzed on Western Blots using antibodies against the Na$^{+}$/Ca$^{2+}$-K$^{+}$ exchanger and $\alpha$- and $\beta$-subunit of the cGMP gated channel. In the native membrane, virtually all Na$^{+}$/Ca$^{2+}$-K$^{+}$ exchanger could be cross-linked mainly to a 490 kDa product by catalysed disulfide formation with cupric-phenanthroline. Stable crosslinks were also obtained with the thiol-specific reagent N,N'-p-phenylenedimaleimide. Neuraminidase treatment of the heavily glycosylated exchanger and of the 490 kDa crosslink reduced the apparent molecular mass by 50 kDa and 85 kDa, respectively. DL-1,4-Bismaleimido-2,3-butanediol (BMBD), a novel SH-specific and cleavable crosslinker, was used to analyse the cleaved crosslinks in two-dimensional SDS gel electrophoresis. Purification of the BMBD cross-linked exchanger and analysis of the cleaved crosslinks identified homodimers of the exchanger. Higher oligomers were not detected, suggesting that the Na$^{+}$/Ca$^{2+}$-K$^{+}$ exchanger exists as a homodimer in the plasma membrane. No crosslinks of the exchanger were obtained after solubilization in 10 mM CHAPS, but a small percentage of the exchanger was cross-linked, when the exchanger was reconstituted into phosphatidylcholine vesicles. This finding suggests that the exchanger dissociates intomonomers, when solubilized in detergent. SH-specific reagents did not crosslink the $\alpha$-subunit of the cGMP-gated channel, unless it was activated by cGMP. Although the Na$^{+}$/Ca$^{2+}$-K$^{+}$ exchanger did not bind cGMP, it showed additional crosslinks with apparent molecular masses of 330 kDa, 465 kDa and 545 kDa, when the channel was activated before crosslinking. These crosslinks of the exehanger were bound to a calmodulin (CaM)-agarose column, whereas the monomeric exchanger and its [...]

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Contributing Institute(s):

1. Publikationen vor 2000 (PRE-2000)

Research Program(s):

1. 899 - ohne Topic (POF3-899) (POF3-899)

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